Biocoat matrigel invasion chamber assay

In vitro cell invasion of H400 and BICR56 cells was assessed at 22 h using a Corning BioCoat Matrigel Invasion Chamber assay (Corning 354480) as previously described^{41 (link)}. Kava constituents FKA (10 μg/ml), FKB (2.5 μg/ml) and yangonin (10 μg/ml) were used to treat the cells in the upper chambers of the assay. Cells were seeded with a density of 3.0 × 10⁵ cells/well. A culture medium with 10% FBS without antibiotics was used in the lower chamber as chemoattractant. After incubation in standard conditions for 22 h, the medium was removed. The cells in the upper chamber were fixed with 100% ice-cold methanol for 2 min at room temperature and stained (UV protected) using 2% crystal violet solution for 2 min, as per manufacturer's instructions. After the removal of the non-migrated cells, the migrated cells (5 random fields/well) were observed under a BH2 Olympus microscope equipped with a PMW-10MD Sony camera. Captured images were analysed to count migrated cells using ImageJ Software (ImageJ v. 1.50i, National Institutes of Health). All the experiments were performed in triplicate.

In vitro cell migration was performed using Thincert Cell Culture Insert For 24 Well Plates (Greiner Bio‐One). Briefly, a volume of 600 µl of cell suspension (1 × 10⁴ cells) in DMEM serum‐free medium was added into the upper chambers. A volume of 600 µL DMEM medium with 10% FBS without antibiotic was added in the lower chamber to induce cell migration. After 24 h incubation, the medium was removed, and migrated cells were fixed with 4% paraformaldehyde and stained using 0,5% crystal violet solution. The non‐invading cells were removed from the insert's upper surface using a cotton swab, and five random fields were photographed using the inverted microscope IX71 system (Olympus). Images were later processed, quantified and analysed using ImageJ software.
In vitro cell invasion was conducted using BioCoat Matrigel Invasion Chamber assay (Corning). The invasion chamber was removed from the freezer and rehydrated with DMEM medium at 37°C. DMEM was added to the insert's interior and the bottom of wells 2 h before plating the cells. The following steps were performed as described above for the migration assay. All the experiments were performed in triplicate.